Optical flow cytometry system

ABSTRACT

Techniques are disclosed relating to fluorescence-based flow cytometry. A flow cytometer may include a partially-reflective surface configured to reflect a first portion of fluorescent emissions from a sample to a first optical sensor and direct a second, greater portion of fluorescent emissions from the sample to a second optical sensor and a controller configured to determine a value representing the intensity of the fluorescent emissions based on a first measurement taken by the first optical sensor, a second measurement taken by the second optical sensor, or both. A flow cytometer may include a baseplate with a first side and a second, opposing side with a flow cell, a laser, and a reflective surface disposed above the first side and an optical sensor and isolating material disposed below the second side. The reflective surface receives fluorescent emissions and reflects at least a portion through the baseplate to the optical sensor. A flow cytometer may include a flow cell, a laser, a first optical sensor positioned to measure scattered laser light, a second optical sensor positioned to measure fluorescent emissions, and a controller configured to adjust the measurements taken by the second optical sensor based on a comparison of measurements taken by the first optical sensor with expected measurements based on a known beam profile of the laser beam.

This application claims the benefit of U.S. Prov. Appl. No. 62/611,847filed on Dec. 29, 2017, which is incorporated by reference herein in itsentirety.

BACKGROUND Technical Field

This disclosure relates generally to systems and methods for performingmeasurements of one or more materials. More particularly, the disclosedsystems and methods relate to flow cytometry technology used to analyzeparticles suspended in a stream of fluid.

Description of the Related Art

Flow cytometry is employed in various biotechnological endeavors such ascell counting, cell sorting, biomarker detection and proteinengineering. Flow cytometry may be used in the clinical diagnosis ofhealth disorders such as blood cancers. Flow cytometry also hasapplications in basic research, clinical practice and clinical trials.Various flow cytometry techniques use light sources to illuminatesamples to be tested and optical sensors to detect resultingfluorescence emitted by the particles.

SUMMARY

In an embodiment, an apparatus comprises a flow cell configured to movea sample including a fluorescent material through the apparatus and afirst light source configured to illuminate the sample in the flow cellto cause the fluorescent material to produce fluorescent emissions. Theapparatus further comprises a first optical sensor including one or morefirst detection cells and a second optical sensor including one or moresecond detection cells, wherein the second detection cells are largerthan the first detection cells. The apparatus also comprises apartially-reflective surface configured to reflect a first portion offluorescent emissions from the sample to the first optical sensor anddirect a second, greater portion of fluorescent emissions from thesample to the second optical sensor. The apparatus includes a controllerconfigured to: receive a first measurement of the first portion offluorescent emissions with the first optical sensor, receive a secondmeasurement of the second portion of fluorescent emissions with thesecond optical sensor, and determine a value representing the intensityof the fluorescent emissions based on the first measurement, the secondmeasurement, or both.

In another embodiment, a method comprises flowing a sample through aflow cell, wherein the sample includes a fluid suspension of one or moreparticles, wherein at least one of the one or more particles includes afluorescent material. The method further comprises illuminating thesample with a first light source as it flows through the flow cell tocause the fluorescent material to produce fluorescent emissions,receiving fluorescent emissions from the sample, and reflecting a firstportion of the fluorescent emissions to a first optical sensor. Themethod also comprises generating, with the first optical sensor, a firstmeasurement of the first portion of the fluorescent emissions from thesample and directing a second, greater portion of the fluorescentemissions to a second optical sensor. The method further comprisesgenerating, with the second optical sensor, a second measurement of thesecond portion of the fluorescent emissions from the sample; anddetermining a value of the intensity of the fluorescent emissions basedon the first measurement, the second measurement, or both.

In still another embodiment, a flow cytometer comprises a flow cellconfigured to move a sample through an examination zone and a laserconfigured to direct a laser beam toward the examination zone to causethe fluorescent material to produce fluorescent emissions. The flowcytometer further comprises a first silicon photomultiplier (SiPM)comprising first detection cells; and a second SiPM comprising seconddetection cells, wherein the second detection cells are larger than thefirst detection cells, wherein the second detection cells are between20-80 percent larger than the first detection cells. The flow cytometeralso comprises a partially-reflective surface configured to reflect afirst portion of fluorescent emissions from the sample to the first SiPMand direct a second, greater portion of fluorescent emissions from thesample to the second SiPM, wherein the ratio of the first portion offluorescent emissions to the second portion of fluorescent emissions isbetween 1:19 and 1:100. The flow cytometer additionally comprises acontroller configured to: receive a first measurement of the firstportion of fluorescent emissions with the first optical sensor, andreceive a second measurement of the second portion of fluorescentemissions with the second optical sensor.

In an embodiment, an apparatus comprises a baseplate including a firstside, a second, opposing side, and an aperture defining an openingthrough the baseplate from the first side to the second side. Theapparatus further comprises a flow cell configured to receive a sample,wherein the flow cell is disposed above the first side and a laserconfigured to direct a laser beam toward the flow cell, wherein thelaser is disposed above the first side. The apparatus additionallycomprises a first reflective surface disposed above the first side, thefirst reflective surface configured to receive light emitted from theflow cell and reflect the light through the opening; a first opticalsensor disposed below the first side, wherein the first optical sensoris configured to receive light reflected by the reflective surface andthrough the opening; and isolating material disposed between the secondside of the baseplate and at least some of the first optical sensor,wherein the isolating material receives the first optical sensor.

In another embodiment, a method comprises flowing a sample through aflow cell, wherein the sample includes one or more particles, wherein atleast one of the one or more particles includes a fluorescent materialand directing laser light at the flow cell at least in part along an xaxis, wherein the wavelength of the laser light is selected to causefluorescent emissions from the fluorescent material. The method furthercomprises receiving, at a reflective surface, fluorescent emissionsemitted at least in part along a y axis from the fluorescent materialand reflecting, with the reflective surface, at least a portion of thereceived fluorescent emissions at least in part along a z axis toward afirst optical sensor. The method also comprises receiving, at the firstoptical sensor, the reflected portion of the fluorescent emissions andmeasuring the intensity of the reflected portion of the fluorescentemissions.

In still another embodiment, an apparatus comprises a flow cellconfigured to receive a fluorescent material, a laser configured to emita laser beam that intersects the flow cell along a first axis andexcites the fluorescent material, and a first optical sensor. Theapparatus further comprises a first reflective surface configured to:receive fluorescent emissions from laser-excited fluorescent material,wherein the fluorescent emissions travel from the laser-excitedfluorescent material to the reflective surface at least in part along asecond axis that is orthogonal to the first axis, and reflect at least afirst portion of the fluorescent emissions toward the first opticalsensor, wherein the reflected fluorescent emissions travel from thefirst reflective surface to the first optical sensor at least in partalong a third axis that is orthogonal to the first axis and the secondaxis; wherein the first optical sensor is configured to measure theintensity of the fluorescent emissions.

In an embodiment, an apparatus comprises a flow cell configured to flowa sample through the apparatus, wherein the sample includes one or moreparticles, wherein at least one of the one or more particles includes afluorescent material. The apparatus further comprises a laser configuredto illuminate the sample with a laser beam, a first optical sensorpositioned to measure a first portion of the laser beam scattered by thesample, and a second optical sensor positioned to measure fluorescentemissions from the sample produced as a result of the sample absorbing asecond portion of the laser beam. The apparatus also comprises acontroller configured to adjust measurements taken by the second opticalsensor based on a comparison of measurements taken by the first opticalsensor with expected measurements based on a known beam profile of thelaser beam.

In another embodiment, an apparatus comprises a flow cell configured toflow a sample through the apparatus, wherein the sample includes one ormore particles, wherein at least one of the one or more particlesincludes a fluorescent material and a laser configured to illuminate thesample with a laser beam. The apparatus further comprises a firstoptical sensor configured to measure signals from the laser that arescattered at an X degree angle relative to the line defined by the laserbeam as it illuminates the sample, wherein X is between 1 and 15degrees; and a second optical sensor configured to measure fluorescentemissions from the sample at a Y degree angle relative to the linedefined by the laser beam as it illuminates the sample, wherein Y isbetween 80 and 100 degrees. The apparatus also comprises a controllerconfigured to adjust measurements taken by the second optical sensorbased on a comparison of measurements taken by the first optical sensorwith expected measurements based on a known beam profile of the laserbeam.

In yet another embodiment, a method comprises flowing a sample throughan examination zone, wherein the sample includes a fluid suspension ofone or more particles, wherein at least one of the one or more particlesincludes a fluorescent material; and illuminating the sample with alaser beam as it flows through the examination zone to cause thefluorescent material to produce fluorescent emissions. The methodfurther comprises receiving at an X degree angle relative to the linedefined by the laser beam as it illuminates the sample, with a firstoptical sensor, laser light scattered by the sample wherein X is between1 and 15 degrees; and measuring, with the first optical sensor, thereceived scattered laser light. The method also comprises receiving at aY degree angle relative to the line defined by the laser beam as itilluminates the sample, with a second optical sensor, fluorescentemissions from the sample wherein Y is between 80 and 100 degrees; andmeasuring, with the second optical sensor, the received fluorescentemissions. The method further comprises adjusting the measurements fromthe received fluorescent emissions from the second optical sensor basedon a comparison of measurements taken by the first optical sensor withexpected measurements based on a known beam profile of the laser beam.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating an embodiment of a flow cytometerincluding a light source, a partially reflective surface, and at leasttwo optical sensors positioned to measure fluorescent emissions from asample.

FIG. 2 is a block diagram illustrating an embodiment of a flow cytometerincluding a light source, an optical sensor positioned to measurefluorescence emissions, and an optical sensor positioned to measurelight from the light source that has been scattered by a sample.

FIG. 3 is a block diagram illustrating an embodiment of a flow cytometerincluding two light sources and six optical sensors positioned tomeasure fluorescent emissions from a sample.

FIG. 4 is a block diagram illustrating an embodiment of a flow cytometerincluding two light sources, six optical sensors positioned to measurefluorescent emissions from a sample, and an optical sensor positioned tomeasure light from the light source that has been scattered by a sample.

FIG. 5 is a drawing showing a top perspective view of portions of theflow cytometer of FIG. 4.

FIG. 6 is a drawing showing a profile side view of portions of the flowcytometer of FIG. 4.

FIG. 7 is a drawing showing a front view of portions of the flowcytometer of FIG. 4.

FIG. 8 is a drawing showing a top view of portions of the flow cytometerof FIG. 4.

FIG. 9 is a drawing showing a cut-away side view of portions of the flowcytometer of FIG. 4.

FIG. 10 is a drawing showing a cut-away top perspective view of portionsof the flow cytometer of FIG. 4.

FIG. 11 is a drawing showing a perspective view of an optical isolationmaterial using in accordance with the disclosed embodiments.

FIG. 12 is a flow chart illustrating a bifurcated path flow cytometrymethod, in accordance with the disclosed embodiments.

FIG. 13 is a flow chart illustrating a three-dimensional light path flowcytometry method, in accordance with the disclosed embodiments.

FIG. 14 is a flow chart illustrating a beam profile-based adjustmentflow cytometry method, in accordance with the disclosed embodiments.

FIG. 15 is a block diagram of an exemplary computer system, which mayimplement a controller in accordance with the disclosed embodiments.

This disclosure includes references to “one embodiment” or “anembodiment.” The appearances of the phrases “in one embodiment” or “inan embodiment” do not necessarily refer to the same embodiment.Particular features, structures, or characteristics may be combined inany suitable manner consistent with this disclosure.

Within this disclosure, different entities (which may variously bereferred to as “units,” “circuits,” other components, etc.) may bedescribed or claimed as “configured” to perform one or more tasks oroperations. This formulation—[entity] configured to [perform one or moretasks]—is used herein to refer to structure (i.e., something physical,such as an electronic circuit). More specifically, this formulation isused to indicate that this structure is arranged to perform the one ormore tasks during operation. A structure can be said to be “configuredto” perform some task even if the structure is not currently beingoperated. A “controller configured to determine a value representing theintensity of fluorescent emissions” is intended to cover, for example, acomputer system has circuitry that performs this function duringoperation, even if the computer system in question is not currentlybeing used (e.g., a power supply is not connected to it). Thus, anentity described or recited as “configured to” perform some task refersto something physical, such as a device, circuit, memory storing programinstructions executable to implement the task, etc. This phrase is notused herein to refer to something intangible. Thus, the “configured to”construct is not used herein to refer to a software entity such as anapplication programming interface (API).

The term “configured to” is not intended to mean “configurable to.” Anunprogrammed FPGA, for example, would not be considered to be“configured to” perform some specific function, although it may be“configurable to” perform that function and may be “configured to”perform the function after programming.

Reciting in the appended claims that a structure is “configured to”perform one or more tasks is expressly intended not to invoke 35 U.S.C.§ 112(f) for that claim element. Accordingly, none of the claims in thisapplication as filed are intended to be interpreted as havingmeans-plus-function elements. Should Applicant wish to invoke Section112(f) during prosecution, it will recite claim elements using the“means for” [performing a function] construct.

As used herein, the terms “first,” “second,” etc. are used as labels fornouns that they precede, and do not imply any type of ordering (e.g.,spatial, temporal, logical, etc.) unless specifically stated. Forexample, references to “first” and “second” optical sensors would notimply a temporal ordering between the two unless otherwise stated.

As used herein, the term “based on” is used to describe one or morefactors that affect a determination. This term does not foreclose thepossibility that additional factors may affect a determination. That is,a determination may be solely based on specified factors or based on thespecified factors as well as other, unspecified factors. Consider thephrase “determine A based on B.” This phrase specifies that B is afactor is used to determine A or that affects the determination of A.This phrase does not foreclose that the determination of A may also bebased on some other factor, such as C. This phrase is also intended tocover an embodiment in which A is determined based solely on B. As usedherein, the phrase “based on” is thus synonymous with the phrase “basedat least in part on.”

DETAILED DESCRIPTION

This disclosure describes techniques for flow cytometry using variouslight sources and optical sensors. Embodiments of various flowcytometers in various configurations are described in reference to FIGS.1-4. FIG. 1 relates to various embodiments in which a flow cytometerincludes reflective surfaces that bifurcate fluorescent emissions. FIG.2 relates to various embodiments in which a flow cytometer is configuredto make beam profile-based adjustments to measurements of fluorescentemissions. FIG. 3 relates to various embodiments in which a flowcytometer includes reflective surfaces that bifurcate fluorescentemissions and two light sources. FIG. 4 relates to various embodimentsin which a flow cytometer includes reflective surfaces that bifurcatefluorescent emissions and two light sources and is configured to makebeam profile-based adjustments to measurements of fluorescent emissions.Embodiments of some of the various components of FIG. 4 are shown inthree-dimensions and described in further detail in reference to FIG.5-11. An exemplary bifurcated path flow cytometry method is describedwith reference to FIG. 12. An exemplary three-dimensional light pathflow cytometry method is described with reference to FIG. 13. Anexemplary beam profile-based adjustment flow cytometry method isdescribed with reference to FIG. 14. Finally, an exemplary computersystem, which may implement the controller described in FIG. 1-14, isdescribed with reference to FIG. 15.

Exemplary Flow Cytometer with Bifurcation

Referring now to FIG. 1, a block diagram illustrating a flow cytometer100 is shown. In various embodiments, flow cytometer 100 includes alight source 110, a reflective surface 140, a first optical sensor 150,and a second optical sensor 152. Light source 110 is configured toilluminate a sample including a fluorescent material in examination zone120 resulting in fluorescent emissions 130. In various embodiments,light source 110 illuminates the sample in examination zone 120 with abeam 112. In some of such embodiments, beam 112 is directed and focusedwith optics 114 and 116. In some of such embodiments, optic 114 is amirror that changes the direction of beam 112 and optic 116 is a lens(e.g., a cylindrical lens) that focuses beam 112 before beam 112intersects with examination zone 120. In various embodiments, beam 112reflects off of optic 114 before passing through optic 116, but in otherembodiments beam 112 first passes through optic 116 before beingreflected off of optic 114. In various embodiments, flow cytometer 100includes additional optics (not shown) configured to manipulate, focus,diffuse, shape, reflect, refract, etc. beam 112.

Fluorescent emissions 130 are emitted from the sample as it isilluminated by light source 110. For simplicity, FIG. 1 showsfluorescent emissions 130 traveling along a horizontal axis becausethese fluorescent emissions 130 are the ones that might be measured byoptical sensors 150 and 152, but it will be understood that fluorescentemissions may radiate in any of a number of directions. In variousembodiments, fluorescent emissions 130 travel from the examination zone120 to the reflective surface 140. In such embodiments, reflectivesurface 140 is configured to reflect a first portion of fluorescentemissions 130 to first optical sensor 150 and direct a second, greaterportion of fluorescent emissions 130 to second optical sensor 152. Invarious embodiments, flow cytometer 100 includes a second reflectivesurface 142 configured to direct at least some of the second portion offluorescent emissions 130 to second optical sensor 152. In someembodiments, second reflective surface 142 is a mirror and reflects all(or all but a negligible portion) of the light that it receives. Inother embodiments, second reflective surface 142 may also be a partiallyreflective surface (embodiments in which second reflective surface 142is a partially reflective surface are discussed herein in connection toFIGS. 3 and 4). In addition to fluorescent emissions 130, side scatteredlight from beam 112 may also travel along the same horizontal axis. Asdiscussed herein, one or more of the optical sensors 150, 152, 320, 322,324, and 326 may be configured to measure side scattered light from beam112.

First optical sensor 150 includes a plurality of detection cells. Secondoptical sensor 152 include a plurality of detection cells that arelarger than the detection cells of first optical sensor 150. Both firstoptical sensor 150 and second optical sensor 152 are configured toreceive fluorescent emissions 130 and measure the portions offluorescent emissions 130 received. First optical sensor 150 and secondoptical sensor 152 are configured to communicate (e.g., through wired orwireless transmission) these measurements to a controller (not shown) asdiscussed herein.

Flow cytometer 100 includes a controller (not shown) configured toreceive a measurement of the first portion of fluorescent emissions 130from first optical sensor 150, receive a measurement of the secondportion of fluorescent emissions 130 from second optical sensor 152, anddetermine a value representing the intensity of fluorescent emissions130 based on the first measurement, the second measurement, or both.

By using the reflective surface 140 to split light between the firstoptical sensor 150 and the second optical sensor 152, the dynamic rangeof fluorescent emissions 130 that flow cytometer 100 is able to detectis broader than the detectable dynamic range of a single optical sensor.In some of such embodiments, the detectable dynamic range of intensityof fluorescent emissions of the flow cytometer 100 is at least sixdecades. As discussed herein, this is because different configurationsof optical sensors (e.g., SiPMs with higher density APD arrays withsmaller individual APDs, SiPMs with lower density APD arrays with largerindividual APDs) may be used together, and a controller may be used tocalculate the intensity of the fluorescent emissions 130 based on themeasurements taken by the first optical sensor 150 and/or themeasurements taken by the second optical sensor 152.

In embodiments, flow cytometer 100 can (e.g., using a controller asdiscussed herein) count a low intensity of fluorescent emissions 130using the measurement taken by the second optical sensor 152 with littleor no weight given to the measurement taken by the first optical sensor150 (e.g., because the first optical sensor will not receive and measuremany photons). Conversely, flow cytometer 100 can (e.g., using acontroller as discussed herein) count a high intensity of fluorescentemissions 130 using the measurement taken by the first optical sensor150 with little or no weight given to the measurement taken by thesecond optical sensor 152 (e.g., because the second optical sensor 152is saturated). As used herein, a “high intensity” of florescent emission130 includes fluorescent emission 130 sufficient to saturate opticalsensor 152. And in cases where the intensity of the fluorescentemissions 130 is in between the low level and the high level, flowcytometer 100 may (e.g., using a controller as discussed herein) use themeasurements taken by first optical sensor 150 and second optical sensor152 in determining the intensity of fluorescent emissions 130 (andtherefore the presence or not a particular protein, compound, chemical,etc.) by, for example calculating a weighted sum. In variousembodiments, the measurement taken by the second optical sensor 152 is adigital measurement (as defined herein) of fluorescent emissions 130 andthe measurement taken by the first optical sensor 150 is an analogmeasurement (also as defined herein) of fluorescent emissions 130.However, in various embodiments, second optical sensor 152 may take bothan analog measurement and a digital measurement. In various embodiments,using the measurements taken by the first optical sensor 150 and secondoptical sensor 152 as discussed herein, the controller of flow cytometer100 is able to determine a value representing the intensity offluorescent emissions 130. In various embodiments, this value may bebased in part on one or more of the analog measurements generated by thefirst optical sensor 150, the digital measurement generated by thesecond optical sensor 152, and an analog measurement of the currentgenerated as a result of photons being absorbed by the second opticalsensor 152. Additionally, in various embodiments, flow cytometer 100 isconfigured to invalidate measurements if the measurements taken by firstoptical sensor 150 and second optical sensor 152 do not corroborate eachother (e.g., one indicates a very high signal while the other indicatesno signal) which may, for example, be indicative of a hardware fault.

In an ideal flow cytometer, the detection of any photons by firstoptical sensor 150 or second optical sensor 152 would be indicative ofthe presence of a particular substance in a sample being tested (e.g.,for the presence of a particular molecule, protein, compound, chemical,etc.), but in various embodiments, stray and reflected light may findits way into an optical sensor 150, 152. Accordingly, in embodiments,the detection threshold of a particular sample being tested (e.g., forthe presence of a particular protein, compound, chemical, etc.) is acertain number (or range of numbers) of photons (e.g., between 20-200photons) above zero. The detection threshold may vary depending on theparticular sample being tested (e.g., protein 1 has a detectionthreshold of 40 photons, protein 2 has a detection threshold of 100photons). Accordingly, one goal of the flow cytometer 100 is to detectthe difference between the threshold number of photons, which would meanthat the sample tests positive for the protein, compound, chemical,etc., and a lower number of photons, which would mean that the sampletests negative. In some embodiments, the difference between thedetection threshold value (e.g., 20-200 photons) and the lower value issufficiently large to verify the presence of a single molecule of thefluorophore attached to a microsphere that has been excited by beam 112as the microsphere passes through examination zone 120. In variousembodiments, flow cytometer 100 may be used to test a plurality ofparticles in a sample (e.g., the microspheres discussed herein) andestimate a concentration of the target material (e.g., the particularprotein, compound, chemical, etc.) in the sample based on the totalnumber of photons counted, the mean number of photons counted perparticle of the sample, a number of times the detection threshold hasbeen exceeded, a known concentration/response curve for the sample, etc.or a combination.

Exemplary Flow Cytometer with Beam Profile-Based Adjustment

FIG. 2 is a block diagram illustrating an embodiment of a flow cytometer200 including light source 110, an optical sensor 150 positioned tomeasure fluorescence emissions 130, and an optical sensor 230 positionedto measure light from the light source 110 that has been scattered by asample. FIG. 2 includes light source 110, beam 112, optics 114 and 116,examination zone 120, fluorescent emissions 130, reflective surface 140,and optical sensor 150 as discussed above. FIG. 2 further includesscattered light 210, reflective surface 220, and optical sensor 230.

Scattered light 210 is a portion of beam 112 (e.g., a laser beam emittedby light source 110) that has been forward scattered by a sample (notshown) in examination zone 120. As used herein, the term “forwardscatter” refers to light scattered in the direction that the beam 112propagates and in angles 15 degrees or less to the right or left of thedirection that the beam 112 propagates. In various embodiments, whenbeam 112 illuminates an object (e.g., a microsphere in a sample), aportion of beam 112 may scatter off the object at an acute angle. Asdiscussed herein, the sample will absorb some of beam 112 and as aresult, the fluorescent material in the sample will emit fluorescentemissions 130. As discussed herein, optical sensor 150 is positioned tomeasure the “side scatter” fluorescent emissions 130. As used herein,“side scatter” refers to portions of fluorescent emissions 130 that areemitted at a Y degree angle from the line defined by beam 112 as itilluminates the sample in examination zone 120. In various embodiments,Y is between 80 and 100 degrees. As shown in FIGS. 3 and 4, a pluralityof optical sensors 150, 152, 320, 322, 324, 326 may be used to measureside scatter fluorescent emissions 130 even though only optical sensor150 is shown in FIG. 2.

Optical sensor 230 is positioned to measure scattered light 210. Invarious embodiments, optical sensor 230 is disposed at an X degree angle(indicated in FIG. 2) relative the line defined by beam 112 as itilluminates the sample in examination zone 120. In various embodiments,X is between 1 and 15 degrees. In various embodiments, reflectivesurface 220 is positioned to receive scattered light 210 and direct itto optical sensor 230. While FIG. 2 shows reflective surface 220directing scattered light 210 in a direction parallel to the directionof beam 112 as it is emitted from light source 110, scattered light 210may be directed in any of a number of angles, including through thesurface of the baseplate 520 as shown in FIGS. 5-10.

In embodiments where beam 112 is a laser beam, it will be understoodthat the beam profile of the laser beam is Gaussian. That is, the centerof the laser beam is more intense than the edge of the laser beam. As aresult, the center of the laser beam delivers more energy than the edgeof the beam. Thus, if the center of beam 112 illuminates a sample, thesample will absorb more energy than if the edge of beam 112 illuminatesthe sample. Accordingly, if a sample that includes fluorescent materialis illuminated by the center of beam 112, it is to be expected that theintensity of the resulting fluorescent emissions 130 will be highercompared to the intensity of the resulting fluorescent emissions 130from the same sample being illuminated by the edge of beam 112.

As discussed herein, the intensity of fluorescent emissions 130 dependsin part on the amount of energy delivered by beam 112 to the fluorescentmaterial in the sample. As discussed herein, in various embodiments theexamination zone 120 is very small and the sample flows through theexamination zone 120 quickly such that the amount of time that beam 112illuminates a particular particle of the sample is short. In variousembodiments, this amount of time is between 1 and 20 microseconds. Inembodiments, flow cytometer 200 (e.g., with a controller (not shown)) isable to determine which part of beam 112 illuminated the sample, andbased on the known beam profile of beam 112 flow cytometer 200 (e.g.,with a controller (not shown)) can adjust measurement taken by opticalsensor 150. In various embodiments, this adjustment is based on acomparison of measurements taken by the optical sensor 150 with expectedmeasurements based on a known beam profile of the beam 112. In someembodiments, the comparison indicates a proportional relationshipbetween a measurement of fluorescent emissions 130 taken by opticalsensor 150 and a measurement of scattered light taken by the opticalsensor 230.

In various embodiments, flow cytometer 200 is able to determine (e.g.,with a controller) where beam 112 (e.g., in the center, at an edge)illuminated the sample by analyzing measurements taken by optical sensor230. For example, if the center of beam 112 illuminates the sample, theamount of scattered light 210 measured by optical sensor 230 will belower than the amount of scattered light 210 that would be measured ifthe edge of beam 112 illuminates the sample. This is because when thecenter of beam 112 illuminates the sample, the sample absorbs more ofthe energy of beam 112 (and thereafter emits some fluorescent emissions130) and therefore less light is scattered. Accordingly, in variousembodiments, flow cytometer 200 determines (e.g., with a controller)where in the beam profile beam 112 has illuminated a sample based on theamount of scattered light 210 as measured by optical sensor 230. Invarious embodiments, flow cytometer 200 includes filters (not shown) tofilter out fluorescent emissions 130 such that fluorescent emissions 130are not measured by optical sensor 230.

For example, if a portion of a sample flowing through examination zone120 includes a microsphere with a single molecule of a sample beingtested (e.g., for the presence of a particular protein, compound,chemical, etc.), the positive detection threshold for that sample may bebetween 20-200 photons as counted by optical sensor 150 (or otheroptical sensors 152, 320, 322, 324, 326 as discussed herein). However,if the measurements taken by optical sensor 230 indicates that the edgeof beam 112 illuminated the sample, a measurement of 45 photons, forexample, taken by optical sensor 150 may be adjusted (e.g., bymultiplying be a scaling factor) upward. Similarly, if the measurementstaken by optical sensor 230 indicate that the center of beam 112illuminated the sample, a measurement taken by optical sensor 150 may beadjusted (e.g., by multiplying be a scaling factor) downward. As usedherein, “adjusting measurements” taken by optical sensor 150 includesincreasing or decreasing the measurements taken by optical sensor 150(or other optical sensors 152, 320, 322, 324, 326 as discussed herein),increasing or decreasing the detection threshold, or a combination. Invarious embodiments, flow cytometer 200 adjusts (e.g., with acontroller) measurements taken by the optical sensor 150 by decreasing adetection threshold for the sample when the measurement taken by theoptical sensor 230 is above a first scatter threshold (e.g., apredetermined number of photons indicating that the edge of beam 112illuminated the sample). Similarly, in various embodiments, flowcytometer 200 adjusts (e.g., with a controller) measurements taken bythe optical sensor 150 by increasing a detection threshold for thesample when the measurement taken by the optical sensor 230 is below asecond scatter threshold (e.g., a predetermined number of photonsindicating that the center of beam 112 illuminated the sample). Invarious embodiments, these techniques may be used to adjust measurementstaken by multiple optical sensors. For example, measurements taken byboth optical sensor 150 and optical sensor 152 may be adjusted based onthe same measurements taken by optical sensor 230.

Exemplary Flow Cytometer with Bifurcation and Two Light Sources

FIG. 3 is a block diagram illustrating an embodiment of a flow cytometer300 including two light sources and at least six optical sensorspositioned to measure fluorescent emissions from a sample. Flowcytometer 300 includes the components of flow cytometer 100 as shown inFIG. 1 with the addition of additional reflective surfaces 310, 312,314, 316; additional optical sensors 320, 322, 324, 326; and a secondlight source 330.

Light source 330 is configured to illuminate a sample including afluorescent material in examination zone 120 resulting in fluorescentemissions 130. In various embodiments, light source 330 illuminates thesample in examination zone 120 with a beam 332. In some of suchembodiments, beam 332 is directed and focused with optics 334 and 336.In some of such embodiments, optic 334 is a lens (e.g., a cylindricallens) that focuses beam 112 and optic 336 is a mirror that changes thedirection of beam 332 before beam 332 intersects with examination zone120. In various embodiments, beam 332 reflects off of optic 336 beforepassing through optic 334, but in other embodiments (as shown in FIG. 3)beam 332 first passes through optical 334 before being reflected off ofoptic 336. In various embodiments, light source 330 emits a beam 332that has a different wavelength than beam 112 emitted by light source110. In various embodiments, flow cytometer 300 includes additionaloptics (not shown) configured to manipulate, focus, diffuse, shape,reflect, refract, etc. beam 332.

As discussed herein, in some embodiments, light source 330 is a redlaser and beam 332 is a red laser beam and light source 110 is a greenlaser and beam 112 is a green laser beam. Further, because differentfluorescent materials are excited by difference frequencies of light andbecause, as discussed herein, the sample may include particles withmultiple types of different fluorescent materials (e.g., differentfluorescent dyes, different fluorophores), light source 110 mayilluminate and excite a first fluorescent material and light source 330may illuminate and excite a second, different fluorescent material.Additionally, in various embodiments, a first fluorescent material, whenexcited, emits fluorescent emissions 130 at a first wavelength and asecond fluorescent material, when excited, emits fluorescent emissions130 at a second wavelength. For example, in some embodiments, lightsource 110 illuminates a sample with green light in examination zone 120and a first fluorescent material in the sample emits first fluorescentemissions 130 (e.g., in the yellow-orange part of the spectrum) andlight source 330 illuminates the sample with red light in examinationzone 120 and a second fluorescent material in the sample emits secondfluorescent emissions (e.g., in the dark red-infrared part of thespectrum).

Flow cytometer 300 includes reflective surfaces 140, 142, 310, 312, 314,and 316. In various embodiments, reflective surfaces 140, 142, 312, and314 are partially reflective surfaces and reflective surfaces 310 and316 are not (e.g., reflective surfaces 310 and 316 are mirrors or havemirrored surfaces). In such embodiments, fluorescent emissions 130travel to reflective surface 140 and 312 from examination zone 120.Reflective surface 140 reflects a first portion of the fluorescentemissions 130 it receives toward optical sensor 150 and passes a secondportion toward reflective surface 142. In turn, reflective surface 142reflects a first portion of the fluorescent emissions 130 it receivestoward optical sensor 152 and passes a second portion toward reflectivesurface 310. Reflective surface 310 reflects the fluorescent emissions130 it receives toward optical sensor 320. Similarly, reflective surface312 reflects a first portion of the fluorescent emissions 130 itreceives toward optical sensor 322 and passes a second portion towardreflective surface 314. In turn, reflective surface 314 reflects a firstportion of the fluorescent emissions 130 it receives toward opticalsensor 324 and passes a second portion toward reflective surface 316.Reflective surface 316 reflects the fluorescent emissions 130 itreceives toward optical sensor 326. In various embodiments, flowcytometer 300 includes filters (e.g., green filters and red filters inembodiments where beam 112 is green light and beam 332 is red light) toprevent light from beam 112 and beam 332 from reaching optical sensors150, 152, 320, 322, 324, 326.

Further, in some embodiments, other filters may be used to preventcertain frequencies of fluorescent emissions 130 from reaching everyoptical sensor 150, 152, 320, 322, 324, 326. For example, if beam 112has a first wavelength (e.g., green light) and causes a firstfluorescent material to emit first fluorescent emissions 130 with asecond wavelength (e.g., yellow light) and beam 332 has a thirdwavelength (e.g., red light) and causes a second fluorescent material toemit second fluorescent emissions 130 with a fourth wavelength (e.g.,infrared light), filters may be used to ensure that first fluorescentemissions 130 at the second wavelength are received by optical sensors150, 152, and/or 320 and not optical sensors 322, 324, or 326.Similarly, filters may be used to ensure that second fluorescentemissions 130 at the fourth wavelength are received by optical sensors322, 324, and/or 326 and not optical sensors 150, 152, or 320.

In various embodiment, optical sensors 150, 152, 320, 322, 324, and 326may be configured to detect different wavelengths of light. As discussedherein, side scattered light may travel in the same direction asfluorescent emissions 130 (e.g., at an angle between 80-100 degreesrelative the line defined by beam 112 as it illuminates the sample inexamination zone 120) and one of the optical sensors 150, 152, 320, 322,324, and 326 may be configured to detect side scattered light from beam112. Similarly, another one of the optical sensors 150, 152, 320, 322,324, and 326 may be configured to detect side scattered light from beam332. In such embodiments, some of the remaining optical sensors 150,152, 320, 322, 324, and 326 may be configured to measure fluorescentemissions 130 emitted by a first fluorescent material after beingilluminated by first light source 110 and the rest of the remainingoptical sensors 150, 152, 320, 322, 324, and 326 may be configured tomeasure fluorescent emissions 130 emitted by a second or thirdfluorescent materials after being illuminated by second light source 330as discussed herein.

In various embodiments, flow cytometer 300 is able to measure thesefirst fluorescent emissions 130 (e.g., to evaluate the presence or notof a substance in the sample under test) and to measure the secondfluorescent emissions 130 to determine additional information about thesample. In such embodiments, flow cytometer 300 (e.g., with acontroller) is able to receive a measurement of the second fluorescentemissions 130 from one or more optical sensors 150, 152, 320, 322, 324,326, and based on the measurement of the second fluorescent emissions130, determine to which set of a plurality of sets a portion (e.g., afluorescent microsphere) of the sample belongs. In such embodiments,various particles of the sample comprise beads (e.g., microspheres asdiscussed herein) including a second, third, or even fourth fluorescentmaterial. Each of these beads is a member of a set of beads. A samplemay be comprised of various sets of beads, and in embodiments, thevarious sets of beads are tagged with a particular material (e.g. afluorescent dye). That is, beads in set one include a first kind offluorescent dye, beads in set two include a second kind of fluorescentdye, etc. In these embodiments, when beam 332 emitted by light source330 illuminates a portion of the sample (e.g., a particular bead in thesample) as it passes through the examination zone 120, the secondfluorescent dye becomes excited and emits second fluorescent emissions130. In various embodiments, the wavelength of these second fluorescentemissions 130 depend on the wavelength of beam 332 and the chemicalproperties of the particular kind of fluorescent dye used as the secondfluorescent dye. These second fluorescent emissions are received (andmeasured) by flow cytometer 300 (e.g., with optical sensors 150, 152,320, 322, 324, and/or 326), the flow cytometer 300 is able to determine(e.g., with a controller), of which set of beads the particular bead isa member based on the second fluorescent emissions 130.

In various embodiments, flow cytometer 300 is able to determine (e.g.,with a controller) the wavelength of the second fluorescent emissions130 and determine which particular kind of fluorescent dye is present(and therefore of which set the particular bead is a member). Forexample, if set one includes a first kind of fluorescent dye that emitssecond fluorescent emissions 130 at a first wavelength (e.g., in thedark red part of the spectrum) when illuminated by beam 332 (e.g., redlight) and if set two includes a second kind of fluorescent dye thatemits second fluorescent emissions 130 at a second wavelength (e.g., inthe infrared part of the spectrum) when illuminated by beam 332 (e.g.,red light), if flow cytometer 300 is able to determine that the secondfluorescent emissions are dark red, then flow cytometer 300 is able todetermine that the bead belongs to set one (and not set two).

Additionally or alternatively, the various sets of beads may includesecond fluorescent materials that emit light at different intensitieswhen illuminated by the same wavelength of beam 332. In suchembodiments, flow cytometer 300 is able to differentiate between thevarious sets of beads based at least in part on the intensity of secondfluorescent emissions. For example, if set one includes a first kind offluorescent dye that emits second fluorescent emissions 130 at a firstintensity (e.g., 200-400 photons) when illuminated by beam 332 (e.g.,red light) and if set two includes a second kind of fluorescent dye thatemits second fluorescent emissions 130 at a second intensity (e.g.,100-200 photons) when illuminated by beam 332 (e.g., red light), if flowcytometer 300 is able to determine that the second fluorescent emissions130 have an intensity of about 300 photons, then flow cytometer 300 isable to determine that the bead belongs to set one (and not set two).

Additionally or alternatively, the various sets of beads may includesecond, third, and fourth fluorescent materials in differentconcentrations. When illuminated by beam 332, the second, third, andfourth fluorescent materials emit various amounts of characteristicfluorescent emissions 130 correlated to the concentration of second,third, and fourth fluorescent materials in the set of beads. In some ofsuch embodiments, one of more of the optical sensors 150, 152, 320, 322,324, 326 are primarily used to measure a particular wavelength offluorescent emissions 130 characteristic of one of the second, third,and fourth fluorescent materials. For example, optical sensor 324 may beconfigured to detect wavelengths of fluorescent emissions 130 from thesecond fluorescent material and optical sensor 326 may be configured todetect wavelengths of fluorescent emissions 130 from the thirdfluorescent material. Based on the proportion of fluorescent emissions130 detected by optical sensor 324 and the optical sensor 326, theconcentration of second and third fluorescent material in the beads inthe sample may be estimated and the particular one of the various setsof beads identified.

Exemplary Flow Cytometer with Bifurcation, Two Light Sources, andBeam-Profile Based Adjustment

FIG. 4 is a block diagram illustrating an embodiment of a flow cytometer400 including all of the features described in FIGS. 1-3 including twolight sources, six optical sensors positioned to measure fluorescentemissions from a sample, and an optical sensor positioned to measurelight from the light source that has been scattered by a sample. Flowcytometer 400 includes the components of flow cytometers 100, 200, and300 as shown in FIGS. 1-3, and the flow cytometer 400 is configured toperform the various functions performed by flow cytometers 100, 200, and300. While flow cytometer 400 includes seven reflective surfaces 140,142, 220, 310, 312, 314, 316 and seven optical sensors 150, 152, 230,320, 322, 324, and 326, other embodiments may include more opticalsensors (e.g., with additional sets of reflective surfaces and opticalsensors in line with the optical sensors 150, 152, 320, 322, 324, and326) or fewer optical sensors (e.g., omitting reflective surfaces 310and 316 and optical sensors 320 and 326). Further, while flow cytometer400 includes all of the components of FIGS. 1-3, a flow cytometer couldhave some or all of the various features described herein in othercombinations. For example, a first embodiment of a flow cytometer mayinclude components of FIG. 1 and FIG. 2 without a light source 330, beam332, optic 334, or optic 336. A second embodiment of a flow cytometermay include the components of FIG. 2 and a light source 330, beam 332,optic 334, and optic 336 but not the additional reflective surfaces 142,310, 312, 314, 316 and respective optical sensors 152, 320, 322, 324,326.

The sample used in connection with flow cytometers 100, 200, 300, 400includes a fluid suspension of one or more particles where at least someof the particles include fluorescent material. As described in U.S.Pats. Nos. 5,747,349 and 6,266,354, each incorporated herein byreference, in various embodiments the sample used in connection withflow cytometers 100, 200, 300, 400 includes fluorescent microspheres,which are beads impregnated with a fluorescent dye. Such microspheres(also referred to herein as “beads”) may include microparticles, beads,polystyrene beads, microbeads, latex particles, latex beads, fluorescentbeads, fluorescent particles, colored particles and colored beads. Invarious embodiments, the microspheres serve as vehicles for molecularreactions. In various embodiments. microspheres or beads range indiameter from 10 nanometers to 100 microns and are uniform and highlyspherical. Microspheres for use in flow cytometry may be obtained frommanufacturers, such as Luminex Corp. of Austin, Tex. In some of suchembodiments, surfaces of the microspheres are coated with a tag that isattracted to a receptor on a cell, an antigen, an antibody, or the likein the sample fluid. So, the microspheres, having fluorescent dyes, bindspecifically to cellular constituents. In various embodiments, two ormore dyes are used simultaneously, each dye being responsible fordetecting a specific condition. The light sources 110, 330 excite thefluorescent dye(s), causing the fluorescent dye(s) to emit light thatcan be detected by optical sensors 150, 152, 230, 320, 322, 324, and326.

In various embodiments, flow cytometer 100, 200, 300, 400 include one ormore controllers. Such controllers may be implemented, for example, bythe components discussed herein in connection to FIG. 15. In suchembodiments, this controller(s) is configured to execute instructionsthat cases the flow cytometer 100, 200, 300, 400 to perform the variousactions (e.g., activating and deactivating light sources 110, 330,taking measurements, flowing samples through flow cytometer 100, 200,300, 400) discussed herein. In various embodiments, the controller isdisposed below the baseplate 520 of flow cytometer 100, 200, 300, 400discussed herein. In embodiments, controller receives output signals(e.g., measurements of light) from the one or more optical sensors 150,152, 230, 320, 322, 324, and 326.

In various embodiments, light source 110 is a green laser and beam 112is a green laser beam. In some of such embodiments, flow cytometer 100,200, 300, 400 includes one or more filters (not shown) to filter outgreen light (e.g., scattered off of the sample) from reaching some orall of optical sensors 150, 152, 320, 322, 324, 326. In variousembodiments, light source 330 is a red laser and beam 332 is a red laserbeam. In some of such embodiments, flow cytometer 300, 400 includes oneor more filters (not shown) to filter out red light (e.g., scattered offof the sample) from reaching some or all of optical sensors 150, 152,320, 322, 324, 326. Additionally, in various embodiments, light source110 and/or light source 330 emit their respective beams 112 and 332continuously, but in other embodiments light source 110 and/or lightsource 330 may emit their respective beams 112 and 332 in pulses (e.g.,a pulse lasting about one nanosecond).

As used herein, the term “examination zone” refers to the portion of aflow cytometer where a beam illuminates a sample flowing through theflow cytometer. In various embodiments, the examination zone is aportion of the flow cell 500 discussed herein. The examination zone isgenerally only a slightly larger than the particles comprising thesample. In some embodiments where the microspheres in the sample areabout 5 microns in diameter, the examination zone measures about 20microns long.

In various embodiments, the reflective surfaces 140, 142, 312, and/or314 are partially reflective surfaces. In some of such embodiments, oneor more reflective surfaces 140, 142, 312, and 314 are pieces of glasswith an anti-reflective coating. In some embodiments, such partiallyreflective surfaces reflect a first portion of the fluorescent emissions130 toward an optical sensor (e.g., optical sensor 150, 152, 322, 324)and pass a second, greater portion of the fluorescent emissions 130through the partially reflective surface toward another reflectivesurface (e.g., reflective surfaces 142, 310, 314, 316) that in turnreflects at least some of the second portion toward another opticalsensor (e.g., optical sensor 152, 320, 324, 326). In variousembodiments, the ratio of the first portion of fluorescent emissions 130to the second portion of fluorescent emissions 130 is between 1:19 and1:100. Further, in various embodiments, one or more reflective surfaces140, 142, 312, and 314 are configured to reflect specific ranges ofwavelengths toward its respective optical sensor 150, 152, 322, 324 suchthat its respective optical sensor is illuminated only with light from acertain spectrum. For example, in various embodiments, reflectivesurface 142 is a piece of glass with an anti-reflective coating andoptical sensors 152 and 320 are configured to measure fluorescentemissions 130 (e.g., light in the yellow-orange spectrum) emitted by afluorescent material in the sample after being illuminated by beam 112(e.g., green light).

In various embodiments, some or all of the optical sensors 150, 152,230, 320, 322, 324, and 326 include a light receiving portion (e.g., aportion containing the APD array discussed herein) configured to receivelight and generate measurements and a support portion containingcomponents configured to transmit one or more measurements (e.g., to acontroller of flow cytometer 100, 200, 300, 400), amplify signals (e.g.,with emitter-coupled logic), filter signals, etc. In variousembodiments, some or all of optical sensors 150, 152, 230, 320, 322,324, and 326 may be mounted on the same circuit board.

In various embodiments, some or all of the optical sensors 150, 152,230, 320, 322, 324, and 326 are silicon photomultipliers (“SiPM”) suchas the SiPMs manufactured by SensL Technologies Ltd. In variousembodiments, SiPMs are solid-state single-photon-sensitive devices builtfrom an avalanche photodiode (“APD”) array on common silicon substrate.In various embodiments, the dimensions of the APDs in the APD array mayvary from 10 to 100 micrometers, and their density can be up to 10000per square millimeter. In embodiments, when the APDs receive a photon(e.g., from fluorescent emissions 130) the APD is reverse-biased and theAPD outputs an avalanche current. Measuring the output avalanche currentfrom the APD array can be used, therefore, to measure the measure theintensity of the light being received by the SiPM. A more intense lightmeans more photons are impacting the APD, and potentially more triggeredavalanche current. However, because the APDs operate using avalanchecurrent, once an impacting photon has triggered the avalanche current ina particular APD, that APD is saturated until the current dissipates. Aslong as an APD is saturated, additional impacting photons do not triggeradditional avalanche current. The amount of time an APD takes to clearthe avalanche current and therefore no longer be saturated depends inpart upon the size of the APD. That is, a smaller APD can clear theavalanche current faster than a larger APD. As used herein, the term“detection cells” refers to elements of an optical sensor that receiveslight and responds to it (e.g., by generating a current, generating avoltage). As used herein, the term “detection cells” includes but is notlimited to APDs in an APD array. However, the term detection cells mayalso refer to the components in any of a number of optical sensor thatreceive impacting photons. Such components may work by using thephotoelectric effect for detection (as APDs do), measuring inducedphonon generation, detecting changes in polarization states of suitablematerials, detecting induced photochemical changes in a material,detecting weak interaction effects, etc.

A SiPM with an APD array consisting of relatively smaller APDs arrangedin a higher density is better able to count the number of photons in alower intensity light signal because each photon in the lower intensitylight signal is more likely to be absorbed by an APD and trigger andavalanche current. This is because a higher density APD with smallerindividual APDs array is more likely to have more individual APDs thatare capable of absorbing an incoming photon in part because there aremore APDs in total and because smaller APDs cease being saturated morequickly compared to a lower density APD array with larger individualAPDs. Put another way, a lower density APD array with larger individualAPDs would become saturated more quickly than a higher density APD arraywith smaller individual APDs. In embodiments, individual SiPMs have adetectable dynamic range of between four and five decades. However, asdiscussed herein, using a lower density APD array with larger individualAPDs in conjunction with a higher density APD array with smallerindividual APDs can extend the detectable dynamic range of the flowcytometer 100, 300, 400 such that the flow cytometer 100, 300, 400 has adetectable dynamic range of six decades or more.

In particular, in various embodiments of flow cytometers, such as flowcytometer 100, 300, and 400, a partially reflective surface 140 reflectsa first portion of fluorescent emissions 130 to first optical sensor 150and directs a second, greater portion of fluorescent emissions to thesecond optical sensor 152. If the first optical sensor 150 is a SiPMwith relatively larger APDs arranged in a lower density and the secondoptical sensor 152 is a SiPM with relatively smaller APDs arranged inhigher density, second optical sensor 152 can be used to count photonsfrom lower intensity fluorescent emissions 130 and first optical sensor150 can be used to count photons for higher intensity fluorescentemissions 130. This is because when fluorescent emissions 130 have alower intensity, second optical sensor 152 is more likely to be able tocount the individual photons because the APD array of the second opticalsensor 152 is not saturated. Because first optical sensor 152 onlyreceives the smaller portion of the lower fluorescent emissions, firstoptical sensor 152 is unlikely to detect many photons. However, whenfluorescent emissions 130 have a higher intensity, second optical sensor152 is more likely to become saturated by received photons and unable tocount additional photons. In this case, because first optical sensor 150receives a smaller portion of the photons, first optical sensor 150 ismore likely to be able take a more useful measurement. In some of suchembodiments, the detection cells of the first optical sensor 150comprise an array of X micron square detection cells and the detectioncells of the second optical sensor 152 comprise and array of Y micronsquare detection cells, wherein the ratio of X:Y is between 1.2:1 to1.8:1. In some of such embodiments, X is 35 microns and Y is 20 microns.

In various embodiments the SiPMs includes a plurality of terminalsincluding an anode, a “fast output terminal,” and a cathode (used as areturn current path for the anode and fast output terminal). As usedherein, the fast output terminal refers to a terminal that outputs asignal that is the derivative of the internal fast switching of theSiPM's various individual APDs in response to the detection of a singlephoton by the individual APDs. The signal output by the fast outputterminal is the sum, for the SiPM's APD array, of the derivative of theinternal fast switching of the various individual APDs. As discussedherein, a “digital measurement” taken by an optical sensor 150, 152,230, 320, 322, 324, and 326 includes the signal output by the fastoutput terminal. In various embodiments, such digital measurements are atotal count of the number of photons detected by the optical sensor(e.g., second optical sensor 152) during a period of time. The signaloutput by the anode is the total avalanche current generated by APDarray. As discussed herein, an “analog measurement” taken by an opticalsensor 150, 152, 230, 320, 322, 324, and 326 includes the signal outputby the anode-cathode. In various embodiments, such analog measurementsare the current generated as a result of photons being absorbed by theoptical sensor (e.g., first optical sensor 150). In various embodiments,flow cytometer 100, 200, 300, 400 receives measurements from the fastoutput terminal and the anode from some or all of the optical sensors150, 152, 230, 320, 322, 324, and 326.

Three-Dimensional Drawings of an Exemplary Flow Cytometer

FIGS. 5-10 are drawings showing various views of a flow cytometer 400 ofany of FIG. 4. FIG. 5 shows a top perspective view of flow cytometer400. FIG. 6 shows a profile side view of flow cytometer 400. FIG. 7shows a front view of flow cytometer 400. FIG. 8 shows a top view offlow cytometer 400. FIG. 9 shows a cut-away side view of flow cytometer400.

FIG. 10 shows a cut-away top perspective view of flow cytometer 400.Light sources 110 and 330 are not shown in FIGS. 5-10, but in variousembodiments either or both are part of flow cytometer 400, and beam 112(e.g., emitted by light source 110) is shown in FIGS. 5 and 8. FIGS.5-10 include flow cell 500, reflective surface array 510, and baseplate520. As shown in FIGS. 6, 7, and 9, flow cytometer 400 also includesoptical isolation material 620 (discussed in further detail herein inconnection to FIG. 11). Optical isolation material 620 may be a singlepiece of material as shown in FIGS. 6, 7, and 9, or it may be more thanone piece of material. For example in various embodiments, a piece ofoptical isolation material 620 is disposed between baseplate 520 andoptical sensors 150, 152, and 320 and another piece of optical isolationmaterial 620 is disposed between baseplate 520 and optical sensors 322,324, and 326. In various embodiments, a piece of optical isolationmaterial 620 is disposed between baseplate 520 and optical sensor 230.

While FIGS. 5-10 show various views of flow cytometer 400 andthree-dimensional light paths as discussed herein, various otherembodiments of a flow cytometer may include three-dimensional lightpaths without including all of the components of flow cytometer 400. Forexample, the various flow cytometers 100, 200, and 300 shown in FIGS.1-3 may include three-dimensional light paths (e.g., with variousreflective surfaces disposed above the baseplate reflecting fluorescentemissions 130 or scattered light 210 through the baseplate to opticalsensors disposed below the baseplate),

Flow cell 500 defines the flow path of a sample under test through flowcytometer 400. As discussed herein, in various embodiments, sample is afluid suspension of one or more particles where at least some of theparticles include fluorescent material. In various embodiments, flowcell 500 is a cuvette. Further, in various embodiments, flow cell 500 istransparent and is made out of an optically clear material such asplastic, glass, or fused quartz. In embodiments, the sample is injectedinto, at, or near, the center of flow cell 500 in a process referred inthe art to as “hydrodynamic focusing.” Ideally, the flow cell 500delivers the sample such that the particles including fluorescentmaterial are delivered reproducibly to the center of the examinationzone 120. As discussed herein, in various embodiments, examination zone120 is a portion of flow cell 500. Further, in various embodiments, flowcell 500 is configured to flow the sample through the examination zone120 one particle at a time, which may allow the fluorescence of aparticular particle of the sample to be measured individually. Putanother way, in various embodiments, flow cell 500 is configured suchthat no more than one entire bead of the sample is disposed in theexamination zone 120 at any particular time. In various embodiments,flow cell 500 flows sample from a reservoir located above first side 600to a second location (e.g., a second reservoir) located below secondside 610.

Reflective surface array 510 is an enclosure that receives variousreflective surfaces 140, 142, 310, 312, 314, 316 and holds thereflective surfaces over the various openings 900 through baseplate 520such that fluorescent emissions 130 reflect off of reflective surfaces140, 142, 310, 312, 314, 316, through openings 900, and to the opticalsensors 150, 152, 320, 322, 324, 326. In various embodiments, reflectivesurface array 510 is opaque and sealed to outside light to minimize thenumber of unwanted photons (e.g., photons that are not part offluorescent emissions 130) that reach optical sensors 150, 152, 320,322, 324, 326. In comparison, in some embodiments there is no enclosureover reflective surface 220 (although in other embodiments, there is anenclosure over reflective surface 220). Having no enclosure overreflective surface 220 may, for example, allow more scattered light 210to reach optical sensor 230.

Baseplate 520 is one or more pieces of material that secure the variouscomponents of flow cytometer 400. In various embodiments, baseplate 520is a made of metal, polymer, composite, or a combination. In variousembodiments, baseplate 520 includes one or more light tunnels 522 thatrestrict light coming from examination zone 120 to reflective surfacearray 510. These light tunnels 522 may be made of any of a number ofmaterials, and include a hole through which fluorescent emissions 130can pass but scattered light 210 is unlike to pass. Baseplate 520includes a plurality of openings 900 (also referred to herein asapertures). In various embodiments, baseplate 520 includes a first side600 and a second, opposite side 610. In various embodiments, baseplate520 defines a plane between the first side 600 and the second side 610.Further, in various embodiments, first side 600 and second side 610 areparallel. For the sake of simplicity here, points in space andcomponents closer to first side 600 than second side 610 are referred toas being “above” first side 600 and points in space and componentscloser to second side 610 than first side 600 are referred to as being“below” second side 610, but it will be understood that the baseplate520 and flow cytometer 400 may be oriented in any direction (e.g., thecenter of the Earth does not necessarily have to be below second side610). The various openings 900 allow light (e.g., fluorescent emissions130 and/or scattered light 210) to pass from above first side 600,through baseplate 520, below second side 610, through optical isolationmaterial 620, and to the various optical sensors 150, 152, 230, 320,322, 324, and 326.

In various embodiments, light source 110; beam 112; optic 114; optic116; examination zone 120; reflective surfaces 140, 142, 220, 310, 312,314, and 316; light source 330; beam 332; optic 334; optic 336; flowcell 500; reflective surface array 510, and light tunnel(s) 522 aredisposed above first side 600. In such embodiments, optical sensors 150,152, 230, 320, 322, 324, and 326; optical isolation material 620; andcontroller (not shown) are disposed below second side 610. In suchembodiments, reflective surfaces 140, 142, 220, 310, 312, 314, and 316are disposed above their respective optical sensors 150, 152, 230, 320,322, 324, and 326. In these embodiments, fluorescent emissions 130and/or scattered light 210 begin above first side 600 and are reflectedthrough openings 900 by various reflective surfaces 140, 142, 220, 310,312, 314, and 316. After passing through baseplate 520 and opticalisolation material 620, fluorescent emissions 130 and/or scattered light210 is received by the various optical sensors 150, 152, 230, 320, 322,324, and 326.

In various embodiments, the arrangement of flow cytometer 400 as shownin FIG. 5-10 confers a number of advantages. For example, by having thevarious visible light paths (e.g., beam 112, beam 332, fluorescentemissions 130, scattered light 210) lie along three dimensions, flowcytometer 400 reduces the possibility that unwanted photons (e.g.,photons that are not from fluorescent emissions 130) reach the variousoptical sensors 150, 152, 320, 322, 324, and 326. As shown in FIG. 5,for example, beams 112 and 332 travel along the y-axis before beingreflected by optic 114 and 336 and then travel along the x-axis andilluminate the sample at the examination zone 120. As discussed herein,after the fluorescent material(s) in the sample is excited by beam 112and/or 332, the fluorescent material produces fluorescent emissions 130.Fluorescent emissions 130 may be emitted in any direction, but a portionof the fluorescent emissions 130 travels in a straight line along they-axis through light tunnel 522 and into reflective surface array 510.The various reflective surfaces 140, 142, 310, 312, 314, and 316 reflectrespective portions of fluorescent emissions 130 through respectiveopenings 900 in baseplate 520 along the z-axis. The fluorescentemissions 130 are then in turn received by the various optical sensors150, 152, 320, 322, 324, and 326. Because light path from examinationzone 120 travels through light tunnel 522, reflective surface array 510,and openings 900 and along the y-axis and z-axis, it is unlikely thatmuch if any light from beam 112 or beam 332 or light from outsidesources will be able to travel along the same path that fluorescentemissions 130 travel. Accordingly, the number of photons received byoptical sensors 150, 152, 320, 322, 324, and 326 that is not fromfluorescent emissions 130 is reduced.

Additionally, because the various optical sensors 150, 152, 230, 320,322, 324, and 326 and the controller are located below second side 610of the baseplate 520, the various electrical and/or optical cablescoupling the controller to the optical sensors 150, 152, 230, 320, 322,324, and 326 may be disposed below second side 610 as well. Accordingly,such cables will not obstruct the path of beam 112, beam 332,fluorescent emissions 130, and scattered light 210.

FIG. 11 is a drawing showing a perspective view of optical isolationmaterial 620. In various embodiments, optical isolation material 620comprises a sheet of polymer having one or more apertures 1100. Invarious embodiments, these apertures 1110 receive portions of thevarious optical sensors 150, 152, 230, 320, 322, 324, and 326 (e.g., thelight receiving portion discussed herein). In various embodiments,optical isolation material 620 is opaque to the visible light spectrumand prevents visible light from reaching the light receiving portion ofthe various optical sensor 150, 152, 320, 322, 324, and 326 except forvisible light (e.g., fluorescent emissions 130 or scattered light 210)reflected by the respective reflective surfaces 140, 142, 220, 310, 312,314, and 316. In various embodiments, optical isolation material 620 isa single sheet of material, or it may be in multiple pieces.

Exemplary Methods

FIGS. 12-14 illustrate various flowcharts representing various disclosedmethods implemented with various flow cytometers 100, 200, 300, 400using one or more controllers. Referring now to FIG. 12, a flowchartillustrating an embodiment of a bifurcated path flow cytometry method1200 is shown. In various embodiments, the various actions associatedwith method 1200 are performed with flow cytometers 100, 300, 400 usingone or more controllers. At block 1202, flow cytometer 100, 300, 400flows a sample through a flow cell 500. As discussed herein, in variousembodiments, the sample includes a fluid suspension of one or moreparticles, wherein at least one of the one or more particles includes afluorescent material. At block 1204, flow cytometer 100, 300, 400illuminates the sample with a first light source 110 as it flows throughthe flow cell 500 to cause the fluorescent material to producefluorescent emissions 130. At block 1206, flow cytometer 100, 300, 400receives fluorescent emissions 130 from the sample. At block 1208, flowcytometer 100, 300, 400 reflects a first portion of the fluorescentemissions 130 to a first optical sensor 150. At block 1210, flowcytometer 100, 300, 400 generates, with the first optical sensor 150, afirst measurement of the first portion of the fluorescent emissions 130from the sample. At block 1212, flow cytometer 100, 300, 400 directs asecond, greater portion of the fluorescent emissions 130 to a secondoptical sensor 152. At block 1214, flow cytometer 100, 300, 400generates, with the second optical sensor 152, a second measurement ofthe second portion of the fluorescent emissions 130 from the sample. Atblock 1216, flow cytometer 100, 300, 400 determines a value of theintensity of the fluorescent emissions 130 based on the firstmeasurement, the second measurement, or both.

Referring now to FIG. 13, a flowchart illustrating an embodiment of athree-dimensional light path flow cytometry method 1300 is shown. Invarious embodiments, the various actions associated with method 1300 areperformed with flow cytometers 100, 200, 300, 400 using one or morecontrollers. At block 1302, flow cytometer 100, 200, 300, 400 flows asample through a flow cell 500. As discussed herein, in variousembodiments, the sample includes one or more particles, wherein at leastone of the one or more particles includes a fluorescent material. Atblock 1304. flow cytometer 100, 200, 300, 400 directs laser light (e.g.,beam 112) at the flow cell 500 (e.g., at examination zone 120) at leastin part along an x axis, wherein the wavelength of the laser light isselected to cause fluorescent emissions 130 from the fluorescentmaterial. At block 1306, flow cytometer 100, 200, 300, 400 receives, ata reflective surface 140, fluorescent emissions 130 emitted at least inpart along a y axis from the fluorescent material. At block 1308, flowcytometer 100, 200, 300, 400 reflects, with the reflective surface 140,at least a portion of the received fluorescent emissions 130 at least inpart along a z axis toward a first optical sensor 150. At block 1310,flow cytometer 100, 200, 300, 400 receive, at the first optical sensor150, the reflected portion of the fluorescent emissions 130. At block1310, flow cytometer 100, 200, 300, 400 measures the intensity of thereflected portion of the fluorescent emissions 130.

Referring now to FIG. 14, a flowchart illustrating an embodiment of abeam profile-based adjustment flow cytometry method 1400 is shown. Invarious embodiments, the various actions associated with method 1400 areperformed with flow cytometers 200, 400 using one or more controllers.At block 1402, flow cytometer 200, 400 flows a sample through anexamination zone 120. As discussed herein, in various embodiments, thesample includes a fluid suspension of one or more particles, wherein atleast one of the one or more particles includes a fluorescent material.At block 1404, flow cytometer 200, 400 illuminates the sample with alaser beam (e.g., beam 112) as it flows through the examination zone 120to cause the fluorescent material to produce fluorescent emissions 130.At block 1406, flow cytometer 200, 400 receives at an X degree anglerelative to the line defined by the laser beam as it illuminates thesample, with a first optical sensor 230, laser light scattered (e.g.,scattered light 210) by the sample wherein X is between 1 and 15degrees. At block 1408, flow cytometer 200, 400 measures, with the firstoptical sensor 230, the received scattered laser light. At block 1410,flow cytometer 200, 400 receives at a Y degree angle relative to theline defined by the laser beam as it illuminates the sample, with asecond optical sensor 150, fluorescent emissions 130 from the samplewherein Y is between 80 and 100 degrees. At block 1410, flow cytometer200, 400 measures, with the second optical sensor 150, the receivedfluorescent emissions 130. At block 1412, flow cytometer 200, 400adjusts the measurements from the received fluorescent emissions 130from the second optical sensor 150 based on a comparison of measurementstaken by the first optical sensor 230 with expected measurements basedon a known beam profile of the laser beam.

Exemplary Computer System

Turning now to FIG. 15, a block diagram of an exemplary computer system1500, which may implement the various components of the controller offlow cytometer 100, 200, 300, 400, is depicted. Computer system 1500includes a processor subsystem 1580 that is coupled to a system memory1520 and I/O interfaces(s) 1540 via an interconnect 1560 (e.g., a systembus). I/O interface(s) 1540 is coupled to one or more I/O devices 1550.Computer system 1500 may be any of various types of devices, including,but not limited to, a server system, personal computer system, desktopcomputer, laptop or notebook computer, mainframe computer system, tabletcomputer, handheld computer, workstation, network computer, a consumerdevice such as a mobile phone, music player, or personal data assistant(PDA). Although a single computer system 1500 is shown in FIG. 15 forconvenience, system 1500 may also be implemented as two or more computersystems operating together.

Processor subsystem 1580 may include one or more processors orprocessing units. In various embodiments of computer system 1500,multiple instances of processor subsystem 1580 may be coupled tointerconnect 1560. In various embodiments, processor subsystem 1580 (oreach processor unit within 1580) may contain a cache or other form ofon-board memory.

System memory 1520 is usable to store program instructions executable byprocessor subsystem 1580 to cause system 1500 perform various operationsdescribed herein. System memory 1520 may be implemented using differentphysical memory media, such as hard disk storage, floppy disk storage,removable disk storage, flash memory, random access memory (RAM-SRAM,EDO RAM, SDRAM, DDR SDRAM, RAMBUS RAM, etc.), read only memory (PROM,EEPROM, etc.), and so on. Memory in computer system 1500 is not limitedto primary storage such as memory 1520. Rather, computer system 1500 mayalso include other forms of storage such as cache memory in processorsubsystem 1580 and secondary storage on I/O Devices 1550 (e.g., a harddrive, storage array, etc.). In some embodiments, these other forms ofstorage may also store program instructions executable by processorsubsystem 1580.

I/O interfaces 1540 may be any of various types of interfaces configuredto couple to and communicate with other devices, according to variousembodiments. In one embodiment, I/O interface 1540 is a bridge chip(e.g., Southbridge) from a front-side to one or more back-side buses.I/O interfaces 1540 may be coupled to one or more I/O devices 1550 viaone or more corresponding buses or other interfaces. Examples of I/Odevices 1550 include storage devices (hard drive, optical drive,removable flash drive, storage array, SAN, or their associatedcontroller), network interface devices (e.g., to a local or wide-areanetwork), or other devices (e.g., graphics, user interface devices,etc.). In one embodiment, computer system 1500 is coupled to a networkvia a network interface device 1550 (e.g., configured to communicateover WiFi, Bluetooth, Ethernet, etc.).

Although specific embodiments have been described above, theseembodiments are not intended to limit the scope of the presentdisclosure, even where only a single embodiment is described withrespect to a particular feature. Examples of features provided in thedisclosure are intended to be illustrative rather than restrictiveunless stated otherwise. The above description is intended to cover suchalternatives, modifications, and equivalents as would be apparent to aperson skilled in the art having the benefit of this disclosure.

The scope of the present disclosure includes any feature or combinationof features disclosed herein (either explicitly or implicitly), or anygeneralization thereof, whether or not it mitigates any or all of theproblems addressed herein. Accordingly, new claims may be formulatedduring prosecution of this application (or an application claimingpriority thereto) to any such combination of features. In particular,with reference to the appended claims, features from dependent claimsmay be combined with those of the independent claims and features fromrespective independent claims may be combined in any appropriate mannerand not merely in the specific combinations enumerated in the appendedclaims.

What is claimed is:
 1. An apparatus comprising: a flow cell configuredto move a sample including a fluorescent material through the apparatus;a first light source configured to illuminate the sample in the flowcell to cause the fluorescent material to produce fluorescent emissions;a first optical sensor including one or more first detection cells; asecond optical sensor including one or more second detection cells,wherein the second detection cells are larger than the first detectioncells; a partially-reflective surface configured to reflect a firstportion of fluorescent emissions from the sample to the first opticalsensor and direct a second, greater portion of fluorescent emissionsfrom the sample to the second optical sensor; and a controllerconfigured to: receive a first measurement of the first portion offluorescent emissions with the first optical sensor, receive a secondmeasurement of the second portion of fluorescent emissions with thesecond optical sensor, and determine a value representing the intensityof the fluorescent emissions based on the first measurement, the secondmeasurement, or both.
 2. The apparatus of claim 1, wherein the firstoptical sensor is configured to generate an analog measurement offluorescent emissions and the second optical sensor is configured togenerate a digital measurement of fluorescent emissions.
 3. Theapparatus of claim 2, wherein the analog measurement the first opticalsensor is configured to generate is of a current generated as a resultof photons being absorbed by the first optical sensor.
 4. The apparatusof claim 2, wherein the digital measurement the first optical sensor isconfigured generate is a total count of the number of photons detectedby the first optical sensor during a period of time.
 5. The apparatus ofclaim 2, wherein the value representing the intensity of the fluorescentemissions is based in part on one or more of the analog measurementgenerated by the first optical sensor, the digital measurement generatedby the second optical sensor, and an analog measurement of the currentgenerated as a result of photons being absorbed by the second opticalsensor.
 6. The apparatus of claim 1, wherein the first optical sensorcomprises a first silicon photomultiplier (“SiPM”) and the secondoptical sensor comprises a second SiPM.
 7. The apparatus of claim 6,wherein the first detection cells of the first SiPM comprise an array ofX micron square detection cells, wherein the second detection cells ofthe second SiPM comprise and array of Y micron square detection cells,wherein the ratio of X:Y is between 1.2:1 to 1.8:1.
 8. The apparatus ofclaim 6, wherein the controller is configured to: receive the firstmeasurement from a fast output terminal of the first SiPM, and receivethe second measurement from an anode of the second SiPM.
 9. Theapparatus of claim 1, wherein the ratio of the first portion offluorescent emissions to the second portion of fluorescent emissions isbetween 1:19 and 1:100.
 10. The apparatus of claim 1, wherein thepartially-reflective surface comprises glass with a non-reflectivecoating.
 11. The apparatus of claim 1, wherein the detectable dynamicrange of intensity of fluorescent emissions of the apparatus is at leastsix decades.
 12. The apparatus of claim 1, further comprising: a secondlight source configured to illuminate the sample in the flow cell tocause a second fluorescent material of the sample to produce secondfluorescent emissions; and a third optical sensor configured to measurethe second fluorescent emissions; wherein the controller is furtherconfigured to: receive a measurement of the second fluorescent emissionsfrom the third optical sensor, and based on the measurement of thesecond fluorescent emissions, determine to which set of a plurality ofsets the sample belongs.
 13. A method comprising: flowing a samplethrough a flow cell, wherein the sample includes a fluid suspension ofone or more particles, wherein at least one of the one or more particlesincludes a fluorescent material; illuminating the sample with a firstlight source as it flows through the flow cell to cause the fluorescentmaterial to produce fluorescent emissions; receiving fluorescentemissions from the sample; reflecting a first portion of the fluorescentemissions to a first optical sensor; generating, with the first opticalsensor, a first measurement of the first portion of the fluorescentemissions from the sample; directing a second, greater portion of thefluorescent emissions to a second optical sensor; generating, with thesecond optical sensor, a second measurement of the second portion of thefluorescent emissions from the sample; and determining a value of theintensity of the fluorescent emissions based on the first measurement,the second measurement, or both.
 14. The method of claim 13, wherein thefirst measurement is an analog measurement and the second measurement isa digital measurement.
 15. The method of claim 14, wherein the analogmeasurement indicates a current generated as a result of photons beingabsorbed by the first optical sensor and the digital measurementindicates a total count of the number of photons detected by the firstoptical sensor during a period of time.
 16. The method of claim 14,wherein determining the value of the intensity of the fluorescentemissions is based in part on one or more of the analog measurementgenerated by the first optical sensor, the digital measurement generatedby the second optical sensor, and an analog measurement of the currentgenerated as a result of photons being absorbed by the second opticalsensor.
 17. The method of claim 13, wherein at least some of theparticles comprise beads including a second fluorescent material,wherein each bead is a member of a set of beads, the method furthercomprising: illuminating a particular bead of the sample with a secondlight source to cause second fluorescent emissions from the secondfluorescent material; receiving the second fluorescent emissions fromthe particular bead; and determining of which set of beads theparticular bead is a member based on the second fluorescent emissions.18. The method of claim 13, wherein the first light source is a greenlaser, the method further comprising filtering out green light from thefluorescent emissions.
 19. A flow cytometer, comprising a flow cellconfigured to move a sample through an examination zone; a laserconfigured to direct a laser beam toward the examination zone to causethe fluorescent material to produce fluorescent emissions; a firstsilicon photomultiplier (SiPM) comprising first detection cells; asecond SiPM comprising second detection cells, wherein the seconddetection cells are larger than the first detection cells, wherein thesecond detection cells are between 20-80 percent larger than the firstdetection cells; a partially-reflective surface configured to reflect afirst portion of fluorescent emissions from the sample to the first SiPMand direct a second, greater portion of fluorescent emissions from thesample to the second SiPM, wherein the ratio of the first portion offluorescent emissions to the second portion of fluorescent emissions isbetween 1:19 and 1:100; and a controller configured to: receive a firstmeasurement of the first portion of fluorescent emissions with the firstoptical sensor, and receive a second measurement of the second portionof fluorescent emissions with the second optical sensor.
 20. The flowcytometer of claim 19, further comprising: a baseplate including a firstside, a second, opposing side, and an aperture defining an openingthrough the baseplate from the first side to the second side; whereinthe flow cell, laser, and partially-reflective surface are disposedabove the first side, wherein the first SiPM is disposed below thesecond side, and wherein the partially-reflective surface is orientedsuch that the first portion of fluorescent emissions is reflectedthrough the opening.